This invention relates to a microfluid apparatus, and a miniaturized chemical analysis apparatus. More particularly, the present invention relates to a liquid mixing apparatus and a liquid mixing method for causing small amounts of two or more liquids (may be referred to hereinafter as “reagent solutions”) to converge in a microchannel, thereby mixing the liquids with high efficiency. The present invention relates, particularly, to a microreactor, etc. for producing, for example, a desired reaction product with high efficiency by a chemical reaction between the liquids mixed in the microchannel.
Further, the present invention relates to a chemical analysis system for separating and analyzing an analyte by utilizing the concentration gradient of a denaturant for the analyte. More particularly, the present invention relates to denaturing gradient gel electrophoresis (DGGE) for separating double-stranded nucleic acids (may hereinafter be referred to simply as “nucleic acids”) according to a difference in base sequence with the use of the concentration gradient of a nucleic acid denaturant, a method for forming a denaturing gradient for such a purpose, and a microchip electrophoretic apparatus for performing such DGGE in a microchannel.
Particularly, the present invention relates to a microchip electrophoretic apparatus, etc. which can efficiently mix buffers containing the denaturant in a microchannel and are thus useful for DGGE.
An example of a chemical analysis apparatus having a microchannel structure is a microchip electrophoretic apparatus. Hereinafter, the technical background for the present invention will be described, with a microchip electrophoretic apparatus taken as an example.
Electrophoresis is often used as a means of separating, purifying or analyzing a biopolymer such as a nucleic acid or protein. Electrophoresis is designed to apply a potential to a gel of agarose, polyacrylamide or the like, or a microchannel (capillary), which has been filled with an electrolyte solution, to move charged particles therein and separate particles according to differences in mobility. In electrophoresing double-stranded nucleic acids, the factor that influences mobility is only a molecular weight (the length of a molecule). Thus, they can be separated based on the difference in the molecular weight (length).
As a method for separating double-stranded nucleic acids based on a difference in base sequence, denaturing gradient gel electrophoresis (DGGE) is known. DGGE is used, for example, in detecting the mutation of a nucleic acid, or detecting single nucleotide polymorphism (SNP). In recent years, DGGE has also been used, for example, in the structural analysis of a microbial community which plays an active role in organic waste water and solid waste treatment processes such as the activated sludge process and anaerobic digestion process such as methane fermentation, or in bioremediation which cleans up and restores contaminated soil and groundwater with the use of microorganisms. In the structural analysis of the microbial community, 16S rRNA gene, which is a sequence common to all microorganisms, is often utilized. That is, nucleic acids are extracted from a sample containing a microbial community to be analyzed, and separated by DGGE. The sample to be analyzed by DGGE is an amplification product amplified using primers for PCR which can amplify a sequence portion of the 16S rRNA gene common to microorganisms. With DGGE, nucleic acids originating from main microorganisms constituting the microbial community can be separated on the gel based on differences in the base sequences of the 16S rRNA gene. Among a plurality of lanes on the same gel, nucleic acids located at the same migration distance can be judged to have the same base sequence, so that differences in the constitution of the microbial community to be analyzed can be analyzed. For example, the 16S rRNA gene of a particular microorganism is similarly handled and electrophoresed in a different lane on the same gel. By comparison of the location of its nucleic acid band, it can be determined whether this microorganism is present in the microbial community of interest.
DGGE has the following drawbacks: Since it takes time for gel preparation and electrophoresis, it is not suitable for high throughput (high speed mass treatment) analysis. Since the size of the gel is as large as about 20 cm×about 20 cm×about 1 mm, a large thermostatic chamber is required, making the entire apparatus upsized. Resolution is low compared with microchip electrophoresis.
In preparing a denaturing gradient gel, moreover, a complicated manual procedure is required, and it is difficult to provide a constant denaturing gradient of the gel. Thus, when the structure of the microbial community is analyzed by DGGE, a proper comparison of data between different gels is difficult.
In the early 1990s, Manz proposed the concept of a miniaturized chemical analysis apparatus incorporating all factors necessary for chemical analysis and biochemical analysis onto one chip, so-called micro total analysis system (μTAS). Since then, various types of μTAS's have been developed.
In microchip electrophoresis, which belongs to a field of μTAS, there is used a microchip comprising a substrate having micro-scale channels with a width and a depth of the order of 10 to 100 μm formed thereon by a microfabrication technology, and another substrate bonded onto the substrate. Generally, this microchip is used in measuring the molecular weight of DNA or RNA. In the measurement of the molecular weight of DNA or RNA, the surfaces of the channels are chemically modified to suppress an electroosmotic flow and, in this state, a solution of a polymer matrix such as linear polyacrylamide or hydroxymethylcellulose is filled into the channels to separate DNA or RNA. Conventional agarose gel electrophoresis requires a migration time of 30 minutes to 1 hour. A microchip electrophoretic apparatus, on the other hand, is advantageous in that migration is completed in 10 minutes or less, thus shortening the analysis time, and the amounts of a sample and a reagent consumed are small.
With the aim of further converting a microchip electrophoretic apparatus into μTAS, Ramsey (Domestic Patent Publication (Japanese Translation of PCT application) No. 1998-507516; Patent Document 1) proposes a microchip laboratory apparatus for analyzing or synthesizing a chemical substance. This microchip laboratory apparatus is characterized by simultaneously controlling the potentials of a plurality of reservoirs in order to transport a subject substance from a reservoir filled with the subject substance toward another reservoir. In this apparatus, however, double-stranded nucleic acids are merely separated based on a difference in molecular weight, and double-stranded nucleic acids are not separated based on a difference in base sequence.
Knapp (Domestic Patent Publication No. 2001-521622; Patent Document 2) proposes a method for analysis of nucleic acid and a method for analyzing the melting point of nucleic acid in a microfluidic device. In these methods, a mutation of a nucleic acid sequence is detected with the use of a chemical denaturant concentration gradient and an oligonucleotide probe. However, it is necessary to design, beforehand, an oligonucleotide probe unique to an intended nucleic acid sample. Thus, only a mutation of a known base sequence can be detected, and a mutation of an unknown base sequence cannot be detected.
Bek (Japanese Patent Application Laid-Open No. 1996-261986; Patent Document 3) proposes a liquid mixing method and a liquid mixing apparatus using an electroosmotic flow on a microchip. In this document, only the method and apparatus for liquid mixing on the microchip are provided, there is no mention of a denaturant concentration gradient or separation of double-stranded nucleic acids.
Righetti (Domestic Patent Publication No. 1998-502738; Patent Document 4) provides a method for detecting a point mutation in a double-stranded nucleic acid fragment with the use of a viscous polymer on a microchip. However, a temporal temperature gradient is utilized, and a dedicated computer program for temperature control is required separately.
Baba (Japanese Patent Application Laid-Open No. 2003-66003; Patent Document 5) proposes a microchip electrophoretic apparatus in which a water-soluble polymer solution is filled into microchannels as a running buffer with a concentration gradient. However, what is formed on a microchip is the concentration gradient of the water-soluble polymer. Thus, measurement of the molecular weights of nucleic acids can be made, but double-stranded nucleic acids cannot be separated based on a difference in base sequence. The formation of the polymer concentration gradient, in particular, involves the complicated task of sequentially filling buffer solutions having different polymer concentrations.
As described above, conventional DGGE has drawbacks, such as a complicated experimental procedure, a long time of analysis, unsuitability for high throughput analysis, necessity for a large apparatus, and low resolution compared with capillary electrophoresis. Another drawback is that comparison of data among different gels is difficult in analyzing a microbial community structure by DGGE. Moreover, μTAS's proposed thus far, including microchip electrophoretic apparatuses, concern measurement of the molecular weights of nucleic acids, or separately require a dedicated computer program for temperature control, etc., or necessitate previous designing of an oligonucleotide probe for detection of a particular base sequence and cannot separate unknown double-stranded nucleic acid fragments based on a difference in base sequence.    Patent Document 1: Domestic Patent Publication No. 1998-507516    Patent Document 2: Domestic patent Publication No. 2001-521622    Patent Document 3: Japanese Patent Application Laid-Open No. 1996-261986    Patent Document 4: Domestic Patent Publication No. 1998-502738    Patent Document 5: Japanese Patent Application Laid-Open No. 2003-66003    Patent Document 6: Japanese Patent Application Laid-Open No. 2001-120971